Investigations within this project concern the cell biology of rare human genetic disorders and normal and abnormal intracellular processes. The research goal is to gain insight into changes in molecular function that underlie various genetic metabolic disorders and work towards treatments for these illnesses. The research focuses on three groups of rare disorders: 1. Disorders of sialic acid metabolism. The key enzyme in the sialic acid biosynthesis pathway is UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Dominant mutations in the allosteric site of GNE cause sialuria, characterized by overproduction of sialic acid. Recessive mutations in GNE cause the neuromuscular disorder GNE myopathy, also called hereditary inclusion body myopathy (HIBM). In 2007, we characterized a knock-in GNE myopathy mouse model and demonstrated that N-acetylmannosamine (ManNAc) rescues the phenotype of the homozygous mutant mice and is a promising treatment for human patients (J Clin Inv (2007) 117:1585-1594). Our ManNAc patent (No. 60/932,451) was licensed to a ManNAc manufacturer, New Zealand Pharmaceuticals. And from 2010-2012 preclinical studies were performed with assistance from the NIH-TRND (Therapies for Rare and Neglected Diseases) program, leading to an IND (Investigational New Drug) application and approval from FDA for the use of ManNAc in GNE myopathy patients. In 2011 a natural history study (11-HG-0218, NHGRI) of patients with Hereditary Inclusion Body Myopathy (HIBM) was initiated, and in 2012 a Phase 1 study started (T-HG-0082, NHGRI): A Phase 1 Randomized, Escalating Single-Dose Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of ManNAc in Subjects with Hereditary Inclusion Body Myopathy (HIBM), for both clinical studies, our group coordinates/performs sample collection, storage and research. In the last year, we wrote an extensive review on all aspects of GNE (Ref 6), studied dynamics of GNE isoforms in mice (Ref. 11), and evaluated the use of other sugars in the sialic acid synthesis pathway as alternative therapeutic options to increase sialylation (Ref 3). We study whether other human disorders of hyposialylation exist (other than GNE myopathy) for which ManNAc (or sialic acid, or the GNE gene) can be a potential therapeutic. For these studies we developed a lectin staining panel, which we applied to a variety of unexplained human renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane. Human renal disorders involving glomerular hyposialylation may benefit from ManNAc as a therapeutic agent (under review). We identified a blood-based biomarker for the identification of human hyposialylation disorders (Patent pending: 61/785,094, US application). This last year we also further characterized the adult onset muscle phenotype of our GNE myopathy knock-in mouse model and tested alternative treatments on our murine model, including feeding with sialic acid pathway intermediates (Ref 3) and GNE gene therapy, mostly intravenous delivered embedded in liposomes (Lipoplex) (manuscript in preparation). Our further studies focuson sub-cellular localization and expression levels of GNE and other enzymes in the sialic acid synthesis pathway (Western blotting and real-time quantitative PCR), pathomechanisms of other human hyposialylation disorders and discovery of additional biomarkers that can serve as parameters for sialylation status. 2. Disorders of lysosome-related organelles (LRO) biogenesis. Such disorders include Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome (CHS), Griscelli syndrome, Gray Platelet syndrome (GPS), and other genetically unclassified disorders. Common clinical features are albinism due to defects in melanosomes and prolonged bleeding times due to platelet defects. We investigate known and unknown LRO-disorders-causing genes (by conventional and next generation sequencing techniques), with the goal of better understanding the biology of this group of diseases. To study the effects of LRO-disorders mutations, we perform cell biological studies on patient material (using immuno-fluorescence, immmuno-EM, and live cell imaging) to examine defective intracellular trafficking and sorting of proteins and organelles in cells. Such cells fail to transport certain lysosome-related organelle resident proteins to their correct destinations, and LRO-disorder gene products are generally involved in recognizing the specific vesicles that give rise to LROs. We also catalogue the clinical and genetic characteristics of the distinct subtypes of HPS and related LRO-disorders. This year we identified genes responsible for 3 new HPS subtypes (HPS-8, HPS-9 and HPS-10; Ref 1, and in preparation), wrote a mutation update on DNA variations in oculocutaneous albinism (Ref 9) and we wrote two extensive reviews, one on BEACH domain containing proteins (including LYST and NBEAL2, responsible for CHS and GPS, respectively; Ref 4) and one about Hermansky-Pudlak syndrome for GeneReviews at GeneTests (ref 2). 3. Genetics of Smith-Magenis syndrome (SMS) and related disorders. SMS is a complex neurobehavioral disorder characterized by multiple congenital anomalies, primarily ascribed to a de novo interstitial deletion of 17p11.2. Molecular analysis of SMS patients may shed light on the variable phenotype and genotype-phenotype correlations and possible treatment decisions. The NIH cohort of SMS patients contains patients with the common 3.7 Mb 17p11.2 deletion (n=80), with atypical 17p11.2 deletions (n=24), and non 17p11.2 deleted patients (n= 44). Our whole genome SNP-array analysis on the atypical deletion group identified different 17p11.2 breakpoints that may influence clinical features (manuscript in preparation). SNP-array analysis of the non-deleted cohort identified several novel (micro) deletions or duplications. Our extensive RAI1 gene analysis on the non-deleted subgroup, including mutation analysis and expression studies, identified 10 patients with RAI1 mutations (5 de novo, 5 familial) and described for the first time decreased RAI1 mRNA expression levels not only in patients with the common 17p11.2 deletion but also in RAI1 mutated patients, and in some non-17p11.2 deleted patients.